Automated diagnostic devices based on molecular biological technology are vital for rapidly confirming presence and identity of pathogens. In many cases, detection of the pathogens employs polymerase-chain-reaction (PCR) as such reaction affords very high sensitivity. On the other hand, and especially in the point-of care market, antibody-based methods enjoy relatively high popularity due to their simple use and disposability. However, most antibody-based tests fail to provide a multiplex format, especially where the test is in the point-of care market.
Recently, various microarray tests and test formats have been developed to allow analysis of several ten to several ten thousand analytes in a biological sample. However, as the analyte spot size on the array has become increasingly smaller, optical detection has become more and more complex. Most typically, currently known microarray tests require conventional lab-bench methods that often require whole-day processes and large amounts of user-handling confined to laboratory settings. Moreover, where the array is relatively small, dedicated detection devices (e.g., to detect fluorescence, luminescence, etc.) operate in conjunction with confocal or other magnifying optics, rendering such devices less suitable for field use.
For example, U.S. patent application 2005/031488 teaches an array of isolated proteins immobilized onto a membrane in a dot blot format where the membrane is suspended in a frame for simplified handling. While such array is relatively simple to handle, quantitative analysis is typically not possible and where large numbers of peptides are required, the size of such arrays is prohibitive to high-throughput screening. Still further, such arrays require isolated and purified protein, which adds substantial effort to the construction of the array. To increase density of the array, multiple fibers coated with selected and purified antigens can be bundled and sliced to produce an array as taught in U.S. Pat. No. 6,887,701. Detection in these arrays can use various formats, and is typically done via fluorescence detection. Such array significantly increases the analyte density, however, is typically limited to qualitative detection of reagents. Moreover, the preparation of such arrays is labor intensive and typically requires isolated compounds.
To circumvent problems associated with isolated peptides, individual clones of a nucleic acid library can be expressed in vitro as taught in U.S. application 2004/161748 or in situ as taught in U.S. application 2005/048580. Similarly, epitope arrays can be expressed in vitro as taught in U.S. application 2006/224329. The recombinant protein is then immobilized to the array carrier using a tag or affinity peptide that is fused to the recombinant protein. While such methods significantly simplify array production and increase probe density, various difficulties nevertheless remain. For example, the recombinant protein preparations must be purified and so add substantial effort. Moreover, quantitative detection is typically limited to fluorescence and/or luminescence methods, which require dedicated and expensive devices.
Compounding difficulties with current antibody-test methods is the fact that most or all methods require highly purified protein to be deposited on to a carrier, especially where quantitative analysis of signals is required. Where such carrier is transparent, attachment of unpurified or partially purified proteins typically fail. Alternatively, nitrocellulose membranes may be employed to couple unpurified or partially purified proteins to the carrier. However, under all or almost all circumstances, quantitative analysis of nitrocellulose bound antigens using antibody methods is not possible. Where such quantitative detection was attempted, detection was limited to fluorometric or chmiluminometric detection (see e.g., Schiavini, D. G., et al. (1989). Quantitative Western immunoblotting analysis in survey of human immunodeficiency virus-seropositive patients. J. Clin. Microbiol. 27: 2062-2066; or Feissner, R., et al. (2003). Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes. Anal. Biochem. 315: 90-94).
Therefore, while numerous methods of microarray tests are known in the art, all or almost all of them suffer from one or more disadvantages. Consequently, there is still a need to provide improved devices and methods to simplify and improve quantitative analysis in antibody-based microarray devices.